Hair Analysis Panel Discussion: Section:
Appendix C, Sharon Seidel

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Appendix C
Pre-Meeting Comments

Hair Analysis: Exploring the State of the Science

Sharon Seidel

ATSDR Hair Analysis Workshop - Charge questions:
Topic #1: Analytical Methods.

Atomic absorption spectroscopy (AAS) is commonly used for individual elements, and can now do more than one element at a time. Lead, for example, is commonly measured by graphite furnace AAS. A well-established conventional laboratory with forensic services typically measures individual elements or a small panel of elements in hair for chronic exposure (e.g., first panel - mercury by cold vapor AAS; lead, arsenic, chromium and cadmium by graphite furnace (GF)-AAS; second panel - cadmium, manganese, nickel and thallium, all by GF-AAS). The AAS methods are considered well-established methods. The amount of hair required for either AAS panel (above) is 0.5 gram. Other analytical methods have the potential to measure a number of elements simultaneously, including inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and mass spectroscopy (ICP-MS). Newer ICP-AES instruments can attain a sensitivity equivalent to single element AAS. ICP-MS is a more sensitive method than AES.

In a carefully conducted study, a major research laboratory at the Centers for Disease Control and Prevention (CDC) reported the determination of 28 elements in hair from non-occupationally exposed U.S. populations.¹ These investigators used ICP-AES for all elements except mercury, which was measured with an LDC mercury monitor. The required minimum hair sample weight was 0.5 gram. Miekeley et al. more recently reported results for ICP-MS analysis of a suite of elements from hair in a Brazilian population, with improved sensitivity compared to ICP-AES.² The amount of hair required was approximately 0.3 gram.

Of the 9 commercial "nutritional hair analysis" laboratories currently operating in the United States, 3 indicate that they primarily use ICP-MS, 4 primarily use ICP-AES, and 1 reports use of directly coupled plasma (DCP)-AES. DCP-AES is an older technique that is potentially less stable than ICP-AES. On average, these laboratories measure 26 elements per hair sample. Nutritional hair analysis laboratories require between 0.3 and 1 gram for the AES methods, and 0.25-1 gram for ICP-MS. Puchyr et al. also discuss preparation of hair for elemental analysis by ICP-MS from a nutritional hair analysis laboratory perspective. ³

Other investigative techniques for measuring elements in hair are reported in the scientific literature. A general discussion of common methods is provided by Jacobs and by Haraguchi et al. ^4,5 Various other methods and example references, e.g: Differential pulse voltametric (DPV); ⁶ Instrumental Neutron Activation Analysis (INAA); ^7,8 Microwave-Induced Plasma Mass Spectroscopy (MIP-MS); ⁹ Capillary electrophoresis (CE) and High Performance Liquid Chromatography (HPLC); ¹⁰ and Particle Induced X-ray Emission (PIXE).¹¹

Laboratory variability has been investigated for the commercial "nutritional hair analysis" laboratories on several occasions.^2,12-14 Inter-laboratory variability was high for reference ranges, results, interpretations and health advice. For example, for one hair sample that was split and sent to six of the laboratories, there was a difference of an order of magnitude or more between laboratories in reported results for over 10 elements, including arsenic, lead, and mercury.¹³ In the same split hair sample, no two laboratories flagged the same element as high, and laboratories had conflicting health interpretations and dietary recommendations based on their analysis of the sample. When intra-laboratory variability was investigated for nutritional hair analysis laboratories, results were similarly discrepant.¹²

Topic #2: Factors Influencing the Interpretation of Analytical Results.
A.) Sample collection and analysis:
Sample collection and preparation methods can have a significant impact on the data collected. Hopps notes that scalp hair has about 90% of follicles in the growth phase at any given time, growing at about 0.45 mm/day.¹⁵ Scalp hair grows in a mosaic pattern over the scalp, with similar growth activity in the various regions of the scalp. However, sampling near the face is usually avoided due to increased likelihood of contamination from sebaceous secretions and facial hygiene products/cosmetics. Miekeley et al. note that larger samples of scalp hair (50 g.), cut into <1cm pieces and manually homogenized, showed homogeneity in repeated analyses of aliquots of the samples.²

Commercial nutritional hair analysis laboratories frequently offer the option of collecting samples of axillary or pubic hair. Hair from these regions of the body grows more slowly, with a much greater proportion in the resting phase, and is likely to be subject to external contamination from apocrine gland secretions, in addition to use of personal hygiene products, clothing, etc. There are no published reference ranges for elements from non-scalp hair. A lack of correlation has been shown between scalp and pubic hair for Ca, Cu, Fe, Mg and Zn.¹⁶

Homogenization can be a concern, particularly if long lengths of hair are collected. Concentrations in hair of a number of environmentally-important elements have been shown to increase from the proximal to distal end of hair, e.g. Pb, Cu, Fe, Mn, and Zn.^17,18 Contamination is also a concern if the laboratory uses a grinding tool that introduces contaminants, as occurred in the preparation of one hair reference material, where Al, Fe, Ti, Mn, and Mg contamination were introduced through use of an agate ball grinding mill.¹⁹

Sample preparation and washing methods vary greatly and can cause different analytical results. Chittleborough provides a detailed review of these issues.²⁰ Various washing recommendations include: no-wash;²⁰ use of a standardized washing procedure recommended by the International Atomic Energy Agency (IAEA) which uses a nonpolar solvent-acetone and deionized water;²¹ a mild ionic detergent-sodium lauryl sulfate emulating a detergent shampoo;¹ and more extreme methods including a chelating agent, EDTA;²² and others (see review by Chittleborough).²⁰ There is no washing method presently available which is capable of reliably removing external contaminants without also affecting endogenously-deposited elements.^20,23-25 While a no-wash approach offers the least disturbance to endogenous elements, the demonstration by scanning electron microscopy of dust, dead skin, etc., adhering to much of the length of unwashed hair samples discourages use of this approach.²⁶ Other aspects of laboratory sample preparation that may be critical include procedures which minimize loss of more volatile elements, such as mercury, during sample dissolution.

A major stumbling block in interpreting metals data for hair is laboratory analytical error. The World Health Organization recommends the following quality assurance methods for laboratory analyses. 1) Reference samples of the same matrix (hair) with known concentrations of the metal (element) should be used as standards. 2) Reference samples should contain the metal (element) at about the same concentration as the samples. 3) If such reference materials are not available, analysis of quality-control samples at different laboratories by different analytical methods must be used (i.e., split samples). 4) Since results may vary over time and for different metals (elements), results should be presented for the corresponding time periods and elements.²⁷

There are various certified reference materials (CRM), for one (mercury) or multiple elements in hair, which meet certification requirements including certified values with a stated level of confidence in each value.^19,28-30 There is no certified hair reference material for all elements currently analyzed by commercial "nutritional hair analysis" laboratories. The Chinese hair CRM, reportedly used by four of these laboratories, certifies 17 elements: Al, As, Ca, Cd, Co, Cr, Cu, Fe, Hg, Mg, Mn, Na, Ni, Pb, Se, Sr, and Zn - about half the elements tested by these laboratories. A common practice among these and other laboratories is to use aqueous element standards, or other non-hair standards such as bovine liver. The difficulty with this is the possibility of complex matrix interferences in the hair sample that are not accounted for by the calibration standard. Reference ranges cited by commercial U.S. nutritional hair analysis laboratories show some rather broad inter-laboratory variations, e.g. arsenic (<0.06 vs. <5 ppm), lead (<0.8 vs. 2-20 ppm), and lithium (0.0035-0.025 vs. 1.25-3 ppm).¹³

Investigations of "nutritional hair analysis" laboratory practices using split samples have shown wide discrepancies.^2,12,13 An approved proficiency testing program for hair element analysis is not available under the Clinical Laboratory Improvement Act (CLIA). This type of analysis is classified as a high-complexity test, with method and accuracy verification left up to the individual laboratory.

"Normal" reference ranges are largely undefined, due to the wide variation in elemental hair concentrations in presumed healthy populations. Contributing factors include geography, age, sex, ethnicity, hair type, hair treatments and other exogenous exposures. Examples of U.S. studies follow. DiPietro et al. published reference intervals for 28 elements in a non-occupationally exposed U.S. adult population.¹ These investigators used extensive questionnaire data to control for many of these factors. A partial list of geometric means for healthy adults in this study includes: arsenic (0.15 ppm); cadmium (<0.15 ppm); nickel (0.39 ppm); and lead (2.43 ppm). A number of population studies have been conducted for mercury in hair. For methylmercury, the geometric mean hair concentration for U.S. women reporting some seafood consumption was 0.36 ppm, and 0.24 ppm for no seafood consumption.³¹ Published clinical references for biomonitoring for metals/elements in hair are sparse. These include arsenic (<1 ppm) and thallium (~5-10 ppb)³² and mercury (<1ppm) and nickel (0.01-1.8 ppm).³³ These are secondary to the established blood and/or urine reference levels, and the problem of external contamination is noted as a major stumbling block which limits the use of the hair references.

Generally speaking the use of the term "normal" is misleading. What is being estimated is a background or baseline level for a population, typically by geographic region, rather than a state of health. Methylmercury data are an exception. Methylmercury exposures commonly occur through consumption of fish and seafood. Clear dose-response relationships have been demonstrated between dietary consumption of mercury-contaminated fish and concentrations in human hair. Methylmercury is the only metal (compound) which has a health benchmark based on hair concentrations. The U.S. EPA has a reference dose (RfD) for methylmercury of 0.0001 mg/kg body wt/day. This is based on a benchmark dose of 11 ppm in maternal hair, equivalent to a maternal blood level of 44 micrograms/L, for developmental neurological abnormalities in infants.³⁴ Several reference range studies for methylmercury are available.^31,35,36

B.) Other factors influencing analytical results:
Hopps notes the sources of elements in hair as: 1) papilla (contacted by blood and lymph) during hair formation; 2) sebaceous glands, sweat glands, desquamating skin cells (endogenous exposures not necessarily related to blood/organ concentrations); 3) and exogenous materials.² Salts of sodium, potassium, and calcium predominate in sweat, but minor amounts of other elements are also found, e.g., zinc.¹⁵ There is evidence for an extra input route from the root sheaths into the hair shaft, other than longitudinal growth, complicating the picture of a simple blood compartment:hair relationship.¹¹ Finally, the lipids and waxes in sebum and skin may contribute to sealing exogenous contaminants into the hair shaft.

Exogenous contaminants can range from personal care products to elements present in air, water, soil, occupational environments, etc. As mentioned above, there is currently no washing method capable of removing exogenous elemental contaminants while leaving endogenous elements undisturbed. Chemicals such as methylmercury, which are generally from dietary sources, suffer less from this drawback, provided unusual sources of inorganic mercury do not complicate the picture, e.g., mercury vapor in occupational settings. Practically speaking, public health concerns are often related to exposure, and hair can serve as an index of overall exposure, if not of biological uptake.

Examples of external contaminants of hair include both personal care products and environmental sources. Hair is a porous material (witness the rapid uptake of water and increase in weight during washing) and may bind through weak ion-exchange sites (e.g., Na, K, Ca, Mg), and through stronger bonds, particularly with sulfur, (e.g, arsenic). Arsenic binds avidly to hair, due to the sulfur content of keratin. Exogenous arsenic is readily taken up by hair and cannot be differentiated from endogenous arsenic.¹⁵ It has been shown that adsorption of other metals such as Al, Cd, Cu, Pb and Zn into scalp hair from aqueous solutions cannot be reversed even by extreme washing methods.²⁵ Hair treatments such as permanents can alter such binding.³⁷ Dandruff shampoos containing selenium can contaminate hair.³⁸ DiPietro et al. noted significant difference between dandruff shampoo vs. regular shampoo for Na, Se, and Ti for men, and between permanents/color and any shampoo for Ba, Ca, Cu, Mg, Na, and Sr for women.¹ Hair dyes may contain metals, e.g., lead in "Grecian Formula."³⁹ Sky-Peck also found that peroxide bleaches and permanents altered S, Ca, Fe, and Ni in hair, peroxide altered Zn, and permanents increased Cu and As concentrations.³⁹

Soil, house dust, and water may contribute contaminants.⁴⁰ Air serves as a contamination source.⁴¹ This is a major concern in occupational settings. Cadmium is an example of a metal where environmental sources contribute to concentrations in hair, e.g., drinking water and dust levels and seasonal influences.⁴²

As noted, gender, ethnicity, diet, age, geographic location, and season are capable of influencing hair reference ranges in populations. Sky-Peck found the following for a healthy midwestern U.S. population: 1) gender – females had higher Ca and Ni and lower Pb, Br and Se compared to males; 2) hair color - blondes had less Fe than brunettes, red-heads had more Fe and Cu; 3) ethnicity/race - Blacks had increased Ca, Fe, Ni, Cr, Mn, As, and Pb, and decreased Hg, compared to Caucasians; Orientals had decreased Ca, Fe, Cu, Mn and Pb; 4) age – a decrease in S, Ca and Sr, and an increase in Pb with age; 5) geography – increased hair strontium in areas with elevated strontium in drinking water, and increased hair lead in industrial/older residential areas.³⁹ Sky-Peck notes that some of the differences in gender and ethnicity may be due to differences in hair treatment and/or environmental exposure. While Sky-Peck found no differences between gray hair and natural hair, other investigators have noted pigmentation effects,⁴³ and it is known that various chemicals, including metals, will bind to melanin.⁴⁴

Other investigators have studied age-related differences in hair elements. Paschal et al. observed age-dependent increases in Ca, Ba, Mg and Sr (Group 2A alkali elements) and Zn up to 12-14 yrs in U.S. residents.⁴⁵ In comparison, an Italian study showed increases in Cu, Zn, Cr and Br, and decreases in Fe, Mn and Sr up to 8 yrs.⁴⁶ In Japanese children, Zn decreased up to 12-14 yrs, and Cu showed a similar trend.⁴⁷ The reason for differences between laboratories and/or populations is not presently known.

Baseline reference values for elements in clinical specimens, including hair, have been referenced by international location.^48,49 International differences are identified for hair Zn, Cd, Cu, Mn and Pb. Some of this geographical difference may be due to differences in environmental metal concentrations, industrialization, etc. Seasonal differences in hair element concentrations, e.g. cadmium, may be due to time spent outdoors and contact with soil, dust, etc.

Topic #3: Toxicological Considerations.

As discussed above, methylmercury is the only element (compound) for which sufficient data exist to define the relationship between concentrations in blood, concentrations in hair, and effects on the target (the developing fetus). It is also the only element (compound) with a health benchmark, the U.S. EPA reference dose, based on a threshold concentration in human hair. It should be noted that this threshold was identified based on massive poisoning incidents in human populations and not on typical (dietary) exposures.^34,50 Forensic medicine has used hair to assess poisoning by other elements, e.g, arsenic and lead. However, these document overwhelming poisoning exposures, rather than a threshold for earliest/most subtle adverse health effects. Nor is there a need in these instances to differentiate between a "normal" background and subtle increases in exposures. Such a distinction is difficult due to the wide variations in background reference ranges. This has caused a number of investigators to conclude that results for an individual are not likely to be meaningful with respect to less drastic environmental/dietary exposures, and that statistical analyses of group data must be employed.^13,42,51,52 Finally, if the goal is also to provide an index of body burden, rather than simply document exposure to environmental contaminants, the lack of a washing technique capable of reliably separating exogenous contaminants from biologically-deposited elements is a substantial concern and must be addressed.

Of the trace elements that have been tested in hair, only a few have research data relating hair concentrations to blood levels and/or tissue concentrations. Aside from mercury, the focus has largely been on aluminum, arsenic, cadmium, chromium, copper, lead, selenium, and zinc. Data highlights are summarized below.

Aluminum (Al) – Aluminum is elevated in hair only in extreme exposures (and even then is inconsistent), and is unrelated to serum or bone aluminum.^53-56 Aluminum dietary intake is unrelated to aluminum in hair, even with controlled dietary intake.⁵⁷ Aluminum in hair is not a useful biological indicator of exposure.

Arsenic (As) – Arsenic is well taken up in hair. Animals show a dose-related increase in hair arsenic.⁴² Forensic hair tests can determine the time-course of chronic arsenic poisoning.⁵⁸ Increased arsenic in soil (<20 to 370 ppm soil As) show a slight correlation with slightly elevated hair arsenic using group statistics (0.02 ppm to 0.06 ppm hair As).⁵⁹ Consumption of drinking water with elevated arsenic concentrations showed a correlation with hair arsenic, using group comparisons.^60-62 This correlation was not seen in a study where drinking water exposure was only modestly above a legal threshold.⁶³ Group statistics show elevated hair As in patients with Blackfoot disease.⁶⁴

Cadmium (Cd) – Animal studies show conflicting results with respect to any correlation between cadmium in hair and the target organ, the kidney.^42,52 The most significant non-occupational exposure to cadmium occurs through tobacco smoke. Smokers have elevated blood cadmium levels compared to nonsmokers. Studies show conflicting results with respect to hair cadmium concentrations in smokers versus non-smokers.^65-67 A nationwide German environmental survey found little correlation with cadmium in hair and active cigarette smoking, although it was the major predictor for blood and urine cadmium concentrations. In contrast, outdoor activities, seasonality, and cadmium in tap water were more important predictors in hair cadmium concentrations, emphasizing the role of exogenous deposition of cadmium into hair.⁶⁷

Copper (Cu) – Taylor's review notes that animal studies showed a proportional relationship between copper in hair and liver.⁵² Yoshinaga et al. found no significant correlation between hair copper and various internal organs, including the liver, in autopsy samples.⁶⁸ Literature studies of human populations show conflicting results with respect to hair versus serum copper.^52,69,70 Serum copper is generally higher in women than in men.^33,49 However, hair copper is inconsistent with respect to sex. Contiera and Folin found no effect of sex on hair copper.⁷¹ Sky-Peck found a modest correlation (p<0.025) for higher hair copper in women compared to men (24 vs. 20 ppm).³⁹ In human patients with biliary cirrhosis, or Wilson's disease (systemic copper intoxication), with increased liver copper, hair copper was typically not increased.⁵² Further studies of Wilson's disease confirmed these findings, with no increase in hair copper in patients with this disease.⁷² In copper deficiencies (malnutrition or Menkes syndrome), hair copper was not significantly reduced.⁵²

Chromium (Cr) – Studies of hair chromium are somewhat limited. A large study (40,872 patients) in England found age-related decreases in hair chromium for males and females [0.98 ppm (mean at age 1-4 yrs) to 0.5 ppm (mean at age 70 plus yrs)], slightly lower hair chromium in males ages 25-49 years, and a correlation between hair and serum chromium, all statistically significant.⁷³ In comparison, a U.S. study found no difference in hair chromium by sex or age in 987 individuals.³⁹ Hair chromium has been hypothesized to increase in gestational diabetes (in early pregnancy), compared to non-diabetic pregnant women.⁷⁴ Hair chromium measurements have been used in monitoring occupational exposures, although blood and urine chromium are the standard biological indices.⁷⁵

Lead (Pb) – There are a number of studies relating lead exposure to tissue concentrations, including hair. Animal studies show a dose-dependent correlated increase in lead in bone and hair during the exposure period.⁷⁶ Isotopic tracer studies have shown the deposition of lead into human facial hair, interpreted as the integral of a blood lead pool over approximately 3 months.⁷⁷ In humans, hair analysis can be used to demonstrate lead poisoning.⁷² Occupational exposures show a correlation between blood and hair lead.^52,78 Lower-level exposures have more variable results,⁵² but larger studies appear to support a relationship between hair and blood lead.⁴² Exogenous deposition of lead onto scalp hair may be influential, e.g., season, dust exposure, and hair treatment.⁴² Centers for Disease Control (CDC) investigators compared hair and blood samples from 189 children to gauge the accuracy of using hair to screen for lead poisoning (mean blood lead 9.8 ug/dl; mean hair lead 7.2 ppm).⁷⁹ Hair lead as a screening method had a 57% sensitivity and 18% false-negative rate. The investigators concluded that hair lead measurements are NOT an adequate method of screening for childhood lead poisoning. The reliable measure of individual lead exposure is a blood lead test.

Selenium (Se) - Animal studies show that: 1) hair selenium is strongly influenced by the chemical form of selenium and the level in the diet, with a greater increase for L-selenomethionine than sodium selenate 2) sodium selenate increases hair selenium but not muscle selenium (the largest body Se pool); and 3) dietary methionine deficiency increases selenium deposition in hair.⁸⁰ These observations suggest caution when evaluating environmental selenium exposures. Population measurements have shown a correlation between low hair selenium and selenium-deficient soils.⁸¹ The hair-to-blood selenium ratio is calculated to be ~3 in dietary selenium deficiency, increasing to 10 as toxic levels are approached. Hair selenium will continue to rise far beyond the plasma saturation concentration, indicating contribution from another body pool.⁸² A hair concentration of >5 ppm Se is reported to be associated with elevated exposure, while a concentration <0.12 ppm Se is reported to be associated with chronic selenium deficiency.⁸³ However, most population studies have preferred blood or urine to indicate selenium exposure.⁸⁴ Exogenous contamination with selenium-containing dandruff shampoos is a serious confounding factor in developed countries.¹ Yoshinaga et al. found no significant correlation between selenium concentrations in hair and in internal organs.⁶⁸

Zinc (Zn) – Zinc in hair has been reviewed by several authors.^52,85-88 These reviewers note that hair is a difficult medium for interpretation of zinc status. The interpretation of zinc concentrations in hair can be obscured by confounders such as sex, body composition, and hair treatment.⁸⁹ In severe zinc deficiency, hair growth slows, producing normal or even elevated hair zinc concentrations.⁸⁷ Yoshinaga et al. found no significant correlation between concentrations of zinc in hair and in various internal organs.⁶⁸ Administration of zinc in the diet did not increase zinc in beard hair.⁹⁰ Serum zinc is typically decreased in dialysis patients. Hair zinc in these patients is not consistent with serum findings.⁹¹

In conclusion, with the exception of methylmercury, there is no good indication that hair analysis offers any improvement over currently available clinical tests to determine individual biological exposure to metals/metalloids of concern.⁹² Occupational texts note that hair analysis is unproven to detect toxic chemicals in the body to account for symptoms and inappropriate in the diagnosis of "environmental" illness.⁹³ Group statistics on hair data, preferably geometric means, may be useful in population screening for exposure to some of these metals (e.g., arsenic). Confounding factors, such as hair treatments, must be controlled for in these studies. Analysis of hair minerals to predict nutritional status is a practice not supported by the state of the science.

Topic #4. Data Gaps and Research Needs.

Generally speaking, further information is needed on concentrations of elements in the hair of individuals with known exposures to trace elements, particularly where environmental exposures are of concern. Laboratory studies of elemental concentrations in blood and target tissues compared to hair concentrations are needed. Such data are important if one is to hypothesize that there is a relationship between hair element concentrations and critical/target organ effects. Clinical studies correlating hair concentrations with clinical conditions (deficiencies or elevations) may also be helpful. Further work is needed on sample washing methods. Standardization on one washing method is important for comparison of studies.

Specific recommendations:

Do not use hair analysis for individual nutritional assessment. The state of the science does not support this application.
If hair analysis is undertaken for comparison of groups, choose element(s) for which the literature supports such an approach, e.g., methylmercury, e.g., NOT aluminum.
When studying control versus exposed groups, chose a group size of sufficient statistical power to determine differences between group means, based on current literature findings.
Use geometric means in analyzing group data.
Collect blood and/or urine samples for comparison with the hair results in the analysis of group data. If this is not feasible for the entire study population, choose a subset of sufficient size to provide statistically meaningful comparison data.
A questionnaire should be administered to each individual in the study, determining: age, sex, ethnicity, hair wash and hair treatment history including products used on hair, swimming habits, time spent outdoors, occupation, smoking history, etc. (e.g, DiPietro et al., 1989).

Topic #5: Identifying scenarios for which hair analysis may be appropriate.

Exposure Scenario	Chemical/ Exposure Pathway	Exposure Chronology	Exposure Duration	Measurable Health Effects (Y/N)
Individual – severe poisoning/forensic Group/population:	Mercury, Arsenic, Lead Methylmercury-diet (fish, seafood) Arsenic, Cadmium, Lead	Past / present Past / present Past / present	Acute (1-2 months min.); chronic Acute (1-2 months min.); chronic Acute (1-2 months min.); chronic	Possible with very high exposure Unlikely unless very high exposure Unlikely unless very high exposure

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